Diversity, taxonomic composition, and functional aspects of fungal communities in living, senesced, and fallen leaves at five sites across North America Jana M. U’Ren1 ,2 and A. Elizabeth Arnold1 ,3 1
School of Plant Sciences, University of Arizona, Tucson, AZ, United States of America Department of Agricultural and Biosystems Engineering, University of Arizona, Tucson, AZ, United States of America 3 Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ, United States of America
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ABSTRACT
Submitted 17 April 2016 Accepted 7 November 2016 Published 13 December 2016 Corresponding author Jana M. U’Ren,
[email protected] Academic editor Francis Martin Additional Information and Declarations can be found on page 24 DOI 10.7717/peerj.2768 Copyright 2016 U’Ren and Arnold Distributed under Creative Commons CC-BY 4.0
Background. Fungal endophytes inhabit symptomless, living tissues of all major plant lineages to form one of earth’s most prevalent groups of symbionts. Many reproduce from senesced and/or decomposing leaves and can produce extracellular leafdegrading enzymes, blurring the line between symbiotrophy and saprotrophy. To better understand the endophyte–saprotroph continuum we compared fungal communities and functional traits of focal strains isolated from living leaves to those isolated from leaves after senescence and decomposition, with a focus on foliage of woody plants in five biogeographic provinces ranging from tundra to subtropical scrub forest. Methods. We cultured fungi from the interior of surface-sterilized leaves that were living at the time of sampling (i.e., endophytes), leaves that were dead and were retained in plant canopies (dead leaf fungi, DLF), and fallen leaves (leaf litter fungi, LLF) from 3–4 species of woody plants in each of five sites in North America. Our sampling encompassed 18 plant species representing two families of Pinophyta and five families of Angiospermae. Diversity and composition of fungal communities within and among leaf life stages, hosts, and sites were compared using ITS-partial LSU rDNA data. We evaluated substrate use and enzyme activity by a subset of fungi isolated only from living tissues vs. fungi isolated only from non-living leaves. Results. Across the diverse biomes and plant taxa surveyed here, culturable fungi from living leaves were isolated less frequently and were less diverse than those isolated from non-living leaves. Fungal communities in living leaves also differed detectably in composition from communities in dead leaves and leaf litter within focal sites and host taxa, regardless of differential weighting of rare and abundant fungi. All focal isolates grew on cellulose, lignin, and pectin as sole carbon sources, but none displayed ligninolytic or pectinolytic activity in vitro. Cellulolytic activity differed among fungal classes. Within Dothideomycetes, activity differed significantly between fungi from living vs. non-living leaves, but such differences were not observed in Sordariomycetes. Discussion. Although some fungi with endophytic life stages clearly persist for periods of time in leaves after senescence and incorporation into leaf litter, our sampling across diverse biomes and host lineages detected consistent differences between fungal assemblages in living vs. non-living leaves, reflecting incursion by fungi from the leaf exterior after leaf death and as leaves begin to decompose. However, fungi found
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How to cite this article U’Ren and Arnold (2016), Diversity, taxonomic composition, and functional aspects of fungal communities in living, senesced, and fallen leaves at five sites across North America. PeerJ 4:e2768; DOI 10.7717/peerj.2768
only in living leaves do not differ consistently in cellulolytic activity from those fungi detected thus far only in dead leaves. Future analyses should consider Basidiomycota in addition to the Ascomycota fungi evaluated here, and should explore more dimensions of functional traits and persistence to further define the endophytism-to-saprotrophy continuum. Subjects Ecology, Microbiology, Mycology Keywords Endophytic fungi, Extracellular enzymes, Diversity, Ascomycota, Plant-fungal
symbioses, Saprotroph
INTRODUCTION Fungal endophytes inhabit symptomless, living photosynthetic tissues of all major plant lineages to form one of earth’s most prevalent groups of symbionts (e.g., Arnold et al., 2010; U’Ren et al., 2012; Zimmerman & Vitousek, 2012; Davey et al., 2013; Balínt et al., 2015). Known from a wide range of biological provinces and agroecosystems, endophytes are a ubiquitous feature of plant biology (e.g., Arnold & Lutzoni, 2007; Saunders, Glenn & Kohn, 2010). Although classified together due to ecological similarities (i.e., colonization and transmission patterns, in planta biodiversity, and host interactions; see Rodriguez et al., 2009), endophytic fungi represent a diversity of evolutionary histories, life history strategies, and functional traits that are only beginning to be understood (reviewed by Porras-Alfaro & Bayman, 2011). Endophytes that inhabit photosynthetic tissues of most plants are horizontally transmitted, form localized infections in aerial tissues, and represent highly diverse and often novel lineages (e.g., Arnold et al., 2009; Gazis et al., 2012; Chen et al., 2015; U’Ren et al., 2016) (Class 3 endophytes sensu Rodriguez et al., 2009; hereafter endophytes). Many reproduce from senesced and/or decomposing leaves (Fröhlich & Hyde, 1999; Promputtha et al., 2007; Promputtha et al., 2010; U’Ren et al., 2010; Chaverri & Gazis, 2011; He et al., 2012). Some also produce extracellular leaf-degrading enzymes (Carroll & Petrini, 1983; Korkama-Rajala, Müller & Pennanen, 2008; Osono & Hirose, 2011; Promputtha et al., 2010; Sun, Guo & Hyde, 2011). Thus many endophytes blur the line between symbiotrophy (during the endophytic phase) and saprotrophy (when they occur in association with dead tissue), creating a challenge for estimating trophic modes and the scale of fungal diversity based on species richness in particular functional groups. The prevalence of saprotrophic life phases among endophytes and the dynamics of such fungi on the endophyte-to-saprotroph continuum are not yet clear. Current evidence suggests that different groups of fungi may persist for longer or shorter periods in senesced leaves, but the relevance of host lineages and abiotic factors is not yet understood in many cases. For example, Osono (2006) estimated that approximately two thirds of fungi with endophytic life stages can persist in and degrade leaf litter. U’Ren et al. (2016) found that 74% of Xylariaceae taxa were represented by isolates from asymptomatic plant tissues, as well as senesced/decomposing leaves, wood, bark, fruits, and/or flowers.
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Some endophytes have ligninolytic and cellulolytic activity, cause mass loss from dead plant tissues in vitro, and persist as litter decomposers over multiple years (e.g., some Lophodermium spp. and xylariaceous fungi; see Koide, Osono & Takeda, 2005; Osono, 2006; Korkama-Rajala, Müller & Pennanen, 2008; Osono & Hirose, 2009; Osono & Hirose, 2011; see also Lindahl et al., 2007; Yuan & Chen, 2014). The presence of such fungi can increase respiration rates and lignocellulolytic activity in litter, altering the litter substrate and the activity of subsequent decomposers (see Koide, Osono & Takeda, 2005; Šnajdr et al., 2011; He et al., 2012; Lin et al., 2015). In contrast, other fungi with endophytic life phases may occupy litter only transiently, quickly sporulating from senesced and decomposing leaves to infect living tissues (e.g., Rhabdocline parkeri on Pseudotsuga menziesii and Coccomyes nipponicum on Camellia japonica; Stone, 1987; Koide, Osono & Takeda, 2005). The impact of such fungi on litter degradation is less well known. Such patterns can be documented via culture-based studies, but also by culture-free methods. For example, fungal genotypes that were dominant in the phyllosphere of living leaves of Quercus petraea disappeared by two to four months post abscission (Voříšková & Baldrian, 2013). Similarly, fungal communities in living leaves of European beech (Fagus sylvatica) in autumn differed from fungi inhabiting leaf litter and bark in the following spring (Unterseher, Peršoh & Schnittler, 2013; but see Peršoh et al., 2013). Together, these studies speak to continuity between fungi that occur within living and non-living leaves (see also Promputtha et al., 2007; Chaverri & Gazis, 2011; Sun, Guo & Hyde, 2011; Peršoh et al., 2013; Voříšková & Baldrian, 2013; Unterseher, Peršoh & Schnittler, 2013). However, it is not clear whether such continuity is consistent in the distinctive plant and fungal communities present in different biomes, nor whether patterns of persistence vary among phylogenetically diverse plants. It is also unclear whether endophytes in a given plant species might occur in non-living leaves of co-occurring plant species: if undetected, such occurrences could alter our understanding of the endophyte–saprotroph continuum. Finally, little is known about functional traits of these fungi, raising the question: do strains that occur only living leaves differ in substrate use and enzyme activity from those that consistently occur in both living and non-living leaves? The goal of this study was to explore the occurrence of endophytes in leaves after senescence and incorporation into the leaf litter, with a focus on woody plants in five biogeographic provinces ranging from tundra to subtropical scrub forest. Our surveys included >7,000 tissue pieces from living leaves, senesced leaves in plant canopies, and recently fallen leaf litter from 3–4 plant species in each site, yielding >2,000 fungal cultures. Here, we address (1) the degree to which fungal communities within leaves differ as a function of leaf type (living, senesced, or in leaf litter) at a given point in time; (2) whether such patterns are consistent among diverse biogeographic/bioclimatic zones and host lineages; and (3) how functional differences in carbon substrate utilization and enzyme activity reflect the occurrence of particular fungi in living vs. non-living leaves.
MATERIALS AND METHODS We collected living leaves, senesced leaves in plant canopies, and leaf litter from five sites representing distinct environmental, biological, and biogeographic regions of
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North America: the Madrean Sky Island Archipelago of southeastern Arizona (AZC); the Appalachian Mountains of western North Carolina (NCH); sub-tropical scrub forest in Florida (FLA); Beringian tundra and boreal forest in the Seward Peninsula ecoregion of western Alaska (AKN); and inland, subalpine tundra in the Interior Highlands of east-central Alaska (AKE) (see U’Ren et al., 2012 for site details). Within each site, we selected three to four species of woody plants that were representative of the community of the region (Table 1; Hultén, 1965; Radford, Ahles & Bell, 1968; Barton, 1994; http://www.archbold-station.org/html/datapub/species/lists/plantlist.html). Overall we examined 18 host species representing two families of Pinophyta and five families of Angiospermae (Table 1). In each site we collected fresh tissues from one individual of each species in each of three replicate microsites located ca. 30 m apart along a 100 m transect, for a total of 9–12 host individuals per site. Within each microsite, focal plants occurred within close proximity to one another (
